The long-term objectives of the proposed research are to understand the molecular basis of photoreceptor disk membrane turnover and some form of photoreceptor degeneration. As a step towards these objectives, the present proposal will investigate: I. The molecular organization of the rod outer segment (ROS) cytoskeleton (i.e. its infrastructure), the actin-based cytoskeleton at the site of disk membrane morphogenesis; II. The properties of ROS calpains (calcium-activated cysteine proteases), which are likely to be involved in the regulation of the cytoskeleton; and III. Degenerative retinas for defects in the cytoskeleton and/or calpain activity. A working hypothesis is that the ROS cytoskeleton has an important role in disk membrane morphogenesis and shedding, and that its breakdown is a critical step in photoreceptor degeneration. These processes might involve proteolysis of components of the ROS cytoskeleton by calpains. Cytoskeletal proteins will be obtained from the detergent-insoluble fraction of isolated ROSs. Actin and microtubule-associated proteins will be identified by their ability to bind to exogenous actin filaments and microtubules. They will be characterized by the conditions of their binding to actin filaments, microtubules, the plasma membrane and each other. Antibodies will be made against the proteins so that their subcellular localization can be determined and to facilitate biochemical characterization. Attention will be paid to the associated proteins that appear most likely to play an important role in disk membrane morphogenesis. Such proteins include those involved in the interactions between the growing end of the actin filaments and the ciliary plasma membrane, or between the actin-myosin contactile system and a fixed point of reference (which is hypothesized to lie with the microtubules of the cilium), or which are substrates for calpains, or whose binding to actin appears to be regulated in a dynamic fashion (by changes in calcium concentration, for example). The sorting of membrane proteins in disk membrane morphogenesis will also be addressed, by examining the distributions of selected ROS disk-specific and ROS plasma membrane-specific proteins at the site of morphogenesis. Retinal calpains will be purified, their activity and cytoskeletal substrates characterized in vitro, and their subcellular distribution determined (especially relative to the cytoskeletal proteins) by using antibodies as probes. Once results have been obtained with normal retinas, examination of a few selected retinal degenerations will be initiated.